BRCA1 AND BRCA2 GENES: ROLE IN HEREDITARY BREASTAND OVARIAN CANCER IN ITALY Manuela SANTAROSA 1 , Riccardo DOLCETTI 1 , Maria Donatella MAGRI 2 , Diana CRIVELLARI 2 , Maria Grazia TIBILETTI 4 , Angelo GALLO 3 , Salvatore TUMOLO 5 , Lara DELLA PUPPA 1 , Daniela FURLAN 4 , Mauro BOIOCCHI 1 * and Alessandra VIEL 1 1 Division of Experimental Oncology 1, Centro di Riferimento Oncologico, Aviano, Italy 2 Prevention, Centro di Riferimento Oncologico, Aviano, Italy 3 Gynecological Oncology, Centro di Riferimento Oncologico, Aviano, Italy 4 Department of Pathology, Ospedale di Circolo, Universita ` dell’Insubria, Varese, Italy 5 Oncologic Unit, Pordenone Hospital, Pordenone, Italy The heritable defects of BRCA1 and BRCA2 genes have been shown to predispose to breast and ovarian cancers. In a previous report, we analyzed 46 Italian families with breast and/or ovarian cancer for BRCA1 mutations. In the present study, those families and 11 others were screened for BRCA2 mutations; the newly enrolled families were also analyzed for the BRCA1 gene. The coding region and splice boundaries of BRCA2 and BRCA1 genes were assessed by the protein- truncation test and single-strand conformational polymor- phism. A total of 20 different mutations were found in 21 families (37%). A total of 9 families (16%) showed mutations in the BRCA1 gene, including the one new mutation identified in this study (5382insC), and 12 families (21%) presented muta- tions in the BRCA2 gene. BRCA2-mutated families presented breast and ovarian cancers or breast cancers only, whereas most BRCA1-mutated families presented ovarian cancer alone or in association with breast cancer. All the BRCA2 mutations led to a truncated protein: 6 were frameshift mutations, 4 were non-sense mutations and 2 involved the intronic invari- ant region leading to splice variants. Therefore, in the Italian population, the cumulative proportion of BRCA1 and BRCA2 mutations was within the range observed in other studies (37%), with higher involvement of BRCA2 than of BRCA1. Many families in which no mutations were found presented a very high incidence of breast and/or ovarian cancer. Among the 36 BRCA1 and BRCA2 wild-type families, 24 presented at least 4 cancer cases, indicating the existence of other impor- tant predisposing genes. Int. J. Cancer 83:5–9, 1999.  1999 Wiley-Liss, Inc. In the last few years, several studies on large families clustering many breast or ovarian cancers have led to the identification of 2 tumor-predisposing genes, BRCA1 and BRCA2 (Miki et al., 1994; Wooster et al., 1995; Tavtigian et al., 1996). Although there are no significant homologies between the BRCA1 and BRCA2 genes, some general similarities exist. Both genes present a huge exon 11. However, in the structure of the BRCA2 gene, exon 10 also is large and, together with exon 11, represents 60% of the total gene. Furthermore, like BRCA1, BRCA2 can be considered a tumor-suppressor gene, since BRCA2- associated tumors frequently present loss of heterozygosity at the BRCA2 locus, due to the loss of the wild-type allele (Collins et al., 1995). Women carrying BRCA1 or BRCA2 mutations have the same high risk (85%) for breast cancer and a 60% and 10% risk, respectively, for ovarian cancer by the age of 70 years (Burke et al., 1997). Furthermore, BRCA2-mutation carriers present a relatively high frequency of male breast cancer (Haraldsson et al., 1998) and have an increased risk of other cancers, such as pancreas cancer (O ¨ zc ¸elik et al., 1997). It has been shown that the actual proportion of families associated with mutations in BRCA1 and BRCA2 is lower than that initially predicted. In fact, the proportion of breast- and ovarian- cancer pedigrees attributable to BRCA1 and BRCA2 ranges from 30% in the British population to 64 to 79% in the populations of the United States, Israel, Iceland and Russia (Szabo and King, 1997). Hitherto no studies on BRCA2 in the Italian population have been reported. We have published our analysis of the BRCA1 mutation spectrum in 46 Italian families in which a low proportion of pathogenetic variants was found (Santarosa et al., 1998). In the present study, we investigated those and other families to assess the proportion of high-risk Italian families presenting mutations in the BRCA2 gene, to determine whether common BRCA2 mutations are present in the Italian population and to compare the prevalence of BRCA1 and BRCA2 mutations in the same group of families. MATERIAL AND METHODS Patients and families We screened 66 patients belonging to 57 families with breast and/or ovarian cancer. The families included fulfilled at least one of the minimum criteria applied in the earlier study (Santarosa et al., 1998). Genomic DNA was purified from blood samples from at least one affected proband, after obtaining informed consent. Mutation screening Mutation screening was performed by single-strand conforma- tional polymorphism (SSCP) and the protein-truncation test (PTT), using the methods described (Santarosa et al., 1998). For BRCA2, 31 primer pairs were used to amplify and analyze all exons (except exons 10 and 11) and exon-intron boundaries (exons 10 and 11 included) by SSCP (Lancaster et al., 1996; Miki et al., 1996; and the Breast-Cancer-Information Core Database). Anneal- ing temperature ranged from 48 to 58°C (details of each primer combination available upon request). Exons 10 and 11 were analyzed by PTT after amplification of one fragment for exon 10 and 5 overlapping segments for exon 11 with the primers used by Foster et al. (1996) and Lancaster et al. (1996). Primers used for BRCA1 analysis have been reported (Santarosa et al., 1998). DNA sequencing All samples that showed an altered SSCP mobility pattern or a shorter protein product were directly sequenced by means of the dye-terminator protocol on an ABI PRISM 310 Genetic Analyzer (Perkin Elmer, Foster City, CA). Reverse-transcription-polymerase-chain reaction (RT-PCR) Total RNA was extracted from lymphocytes using the Triazol reagent for RNA (Life Technologies, Grand Island, NY). First- strand cDNA was synthesized with avian-myeloblastosis-virus reverse transcriptase from 1 μg of total RNA in a total volume of *Correspondence to: Division of Experimental Oncology 1, Centro di Riferimento Oncologico, Via Pedemontana Occidentale 12, 33081 Aviano (PN), Italy. Fax: +39 434 659428. E-mail: mboiocchi@ets.it Received 12 January 1999; Revised 9 April 1999 Int. J. Cancer: 83, 5–9 (1999)  1999 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer